This is in application for the ADAMHA Scientist Develop Award. The long- term objective of this research project is to analyze the kinetic properties of alcohol-metabolizing enzymes. The hypothesis is that human alcohol dehydrogenases vary in kinetic mechanism and rate-limiting step, and that chronic alcohol consumption perturbs the kinetic mechanism of the microsomal ethanol-oxidizing system (MEOS). Ingested alcohols are metabolized predominantly by either alcohol dehydrogenases or MEOS. At least five alcohol dehydrogenase subunits are expressed in the liver and include alpha, beta, and gamma (Class I), eta (Class II), and chi (Class III). A sixth class of alcohol dehydrogenase, sigma, is expressed in the stomach and may be involved in first-pass metabolism. These isoenzymes, with natural substitutions at positions 48 and 93 in the substrate binding site, differ considerably in substrate specificity. Natural substitutions of His-47 or Cys-369 for Arg-47 and Arg-369 in the beta subunit produces isoenzymes that differ dramatically in coenzyme binding affinity. The MEOS is a membrane-bound multi-enzyme complex containing P450 isoenzymes. Long- term alcohol exposure in rats leads to an increase in a hepatic ethanol- induced isoenzyme, P450 IIE, and alters membrane composition in the cells. These changes may affect the kinetic mechanism of the P450 IIE isoenzyme. I have examined by stopped-flow techniques the coenzyme binding characteristics of human beta1beta1 enzymes expressed in E. coli with mutations at position 47. I will use this Scientist Development Award to develop stopped-flow methods for examining the coenzyme and substrate apparent binding rate constants and limiting hydride transfer rates of beta1beta1 enzymes containing site-specific mutations at position 369 and of stomach sigma-alcohol dehydrogenase. I will develop research techniques for using stopped-flow kinetics to study the kinetic mechanism and substrate specificity of P450 IIE, and to determine the effects of long- term alcohol exposure on P450 IIE mechanism in microsomal liposomes. I will also use site-directed mutagenesis and molecular graphics to express and purify enzymes with site-specific mutations at positions 48 and 93, and then with these mutants construct a lattice structure of substrate specificity with molecular graphics.